Electrochemical enzyme assay

ABSTRACT

A diagnostic kit, method, and apparatus for electrochemically determining the presence or concentration of an analyte in a sample. A mixture is formed which includes the sample, an enzyme acceptor polypeptide, an enzyme donor polypeptide, and a labeled substrate. The enzyme donor polypeptide is capable of combining with the enzyme acceptor polypeptide to form an active enzyme complex. The formation of such the active enzyme complex is responsive to the presence or concentration of the analyte in the fluid sample. The active enzyme hydrolyzes the labeled substrate, resulting in the generation of an electroactive label, which can then be oxidized at the surface of an electrode. A current resulting from the oxidation of the electroactive compound can be measured and correlated to the concentration of the analyte in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional of application Ser. No. 08/494,668, filedJun. 26, 1995, which is a continuation-in-part of application Ser. No.08/113,548, filed Aug. 27, 1993, which is now U.S. Pat. No. 5,427,912,issued Jun. 27, 1995.

FIELD OF THE INVENTION

[0002] This invention relates generally to the field of electrochemicalenzyme assays.

BACKGROUND OF THE INVENTION

[0003] Radioimmunoassay was developed in 1960 by Yarlow and Berson as amethod for detecting or quantitating antigens or antibodies usingradiolabled reactants. Since the initial studies in 1960,radioimmunoassay (RIA) has developed into a versatile analyticaltechnique, particularly useful in clinical laboratories to quantitate awide variety of compounds. With RIA, the unknown concentration of anunlabeled antigen is determined by comparing its inhibitory effect onthe binding of a radioactively-labeled antigen to an antibody. RIAs dohave a number of significant limitations, however, including a limitedshelf-life, high cost, and potential environmental harm.

[0004] The disadvantages associated with RIAs led to the development ofthe enzyme immunoassay (EIA), in which the activity of an enzyme ismeasured to quantitate an analyte. EIAs are subdivided intoheterogeneous assays and homogeneous assays. Heterogeneous EIAs requirea physical separation of the antibody-bound, labeled analyte from theunbound labeled analyte. With homogenous EIAs, a separation step is notrequired. Homogeneous EIAs have been successful commercially because oftheir speed, simplicity, and automation. The enzymatic activityassociated with EIAs is often monitored spectrophotometrically, using asubstrate which produces a unique chromophore as a result of anenzymatic reaction.

[0005] In addition to using spectrophotometric detection techniques,EIAs have been developed which use electrochemistry to monitor activityof the enzyme label. With electrochemical detection, the active enzymecauses the formation of an active electron mediator or a redox couplefrom an inactive substrate. The activated mediator or redox couple thenshuttles electrons from the enzyme to the electrode or from theelectrode to the enzyme. The resulting current can be measured andcorrelated to analyte level.

[0006] Direct electrochemical enzymatic assays (non-immunological) arealso known in which the presence or absence of the analyte to bemeasured causes an electroactive compound to be cleaved from anon-electroactive substrate. The electroactive compound may then beoxidized or reduced and the resulting current measured.

[0007] Enzyme complementation immunoassays have also been developed,such as CEDIA® (Cloned Enzyme Donor ImmunoAssay—a registered trademarkof the Microgenics Corporation) technology, an example of which isdescribed in U.S. Pat. No. 4,708,929 (issued Nov. 24, 1987), which ishereby incorporated by reference. CEDIA® technology involves the use ofenzyme acceptor and enzyme donor polypeptides prepared by recombinantDNA techniques or synthetic peptide synthesis techniques which arecapable of spontaneously associating in solution to form an activeenzyme complex. The association can be modulated, for example, byconjugating the enzyme donor polypeptide to a member of a specificbinding pair, and providing the complimentary member of the specificbinding pair elsewhere in the assay. The enzyme donor polypeptide mayalso be chemically modified to include a specific recognition site thatis not a member of a specific binding pair (e.g., a protease site or anesterase site).

[0008] Accordingly, in its broadest sense, CEDIA® technology allows theformation of an active enzyme complex by the spontaneous association ofenzyme acceptor and enzyme donor polypeptides to be dependent on thepresence or concentration of an analyte of interest. The amount ofenzymeatic activity is then monitored spectrophotometrically.

[0009] One embodiment of CEDIA® technology is shown in FIG. 1. Analyteanalog 1 is covalently attached to enzyme donor polypeptide 2 to formenzyme donor polypeptide conjugate 3. Analyte-specific antibody 4 can beused to inhibit reassembly of enzyme donor polypeptide conjugate 3 withenzyme acceptor polypeptide 6. When a sample containing analyte 8 isintroduced, analyte 8 and enzyme donor polypeptide conjugate 3 competefor binding to antibody 4. As the amount of analyte 8 increases, lessenzyme donor polypeptide conjugate 3 binds to antibody 4 and more activeenzyme 10 if formed. Active enzyme 10 hydrolyzes enzyme substrate 11(e.g., chlorophenol-red-β-D-galactopyranose (CPRG)), which thenundergoes a color change and is monitored spectrophotometrically.

SUMMARY OF THE INVENTION

[0010] The present invention is based on the novel combination of CEDIA®technology (i.e., the modulation of enzyme activity in response to thepresence or concentration of an analyte) with electrochemical detectionof the resulting enzyme activity in order to determine the presence orconcentration of the analyte. The advantages that result from thiscombination include the speed and simplicity of a homogeneous EIA andthe simplicity, enhanced analyte sensitivity, small sample volumerequirement, and adaptability to sensor formats associated withelectrochemical measurement of enzyme activity.

[0011] The assay components include an enzyme acceptor polypeptide (EA),an enzyme donor polypeptide (ED), a substrate for enzymatic reaction,and a label which is bound to the substrate and is preferablynonelectroactive until cleaved from the substrate. ED is capable ofcombining with EA to form an active enzyme complex, the formation of theactive enzyme complex being responsive to the presence or concentrationof an analyte in a sample.

[0012] The sample containing the analyte is mixed with a first reagent(EA reagent) which includes the EA. This mixture is then mixed with asecond reagent (ED reagent) which includes ED and the labeled substrate.The enzyme activity resulting from the combination of EA and ED isresponsive to the presence or concentration of the analyte. The activeenzyme then cleaves the label from the substrate, which may be detectedelectrochemically. The current measured from the oxidation of the labelmay then be correlated to the concentration of the analyte in thesample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a block diagram of the assay components of oneembodiment of CEDIA® technology.

[0014]FIG. 2 is a block diagram of one embodiment of the presentinvention.

[0015]FIG. 3 is a representation of the biochemical events of oneembodiment of the present invention, using the example of4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β-D-galactopyranoside as a labeledsubstrate.

[0016]FIG. 4 is a schematic top view of an embodiment of an electrocellused with the present invention.

[0017]FIG. 5 is a schematic cross-sectional view of FIG. 4 taken alongline 5-5 of FIG. 4.

[0018]FIG. 6 is a schematic top view of an embodiment of an immunosensorof the present invention, excluding the fourth insulating substrate.

[0019]FIG. 7 is a schematic cross-sectional view of FIG. 6 taken alongline 7-7 if FIG. 6, including the fourth insulating substrate.

[0020]FIG. 8 is a schematic top view of an embodiment of an immunosensorof the present invention, excluding the fourth insulating substrate.

[0021]FIG. 9 is a schematic cross-sectional view of FIG. 8 taken alongline 9-9 of FIG. 8, including the fourth insulating substrate.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention includes EA and ED which are capable ofspontaneously combining to form an active enzyme complex, wherein suchcombination is responsive to the presence or concentration of an analytein a fluid sample. For example, ED may be modified so that EDspontaneously combines with EA unless hindered by the presence orconcentration of an analyte. Such a modification may be, for example,conjugation of ED to a member of a specific binding pair, wherein theconjugate is capable of spontaneously combining with EA to generateenzyme activity. The member of the specific binding pair conjugated toED may be an analyte or an analyte analog, or a complimentary member ofa specific binding pair (e.g., antibody) which binds to the analyte.

[0023] The EA and ED may be contained in reagents which includecomponents necessary for modulating enzyme activity in response to thepresence or concentration of an analyte. For example, the reagents mayinclude antibodies which bind specifically to the analyte and to analyteconjugated to ED, thereby hindering the spontaneous association of EAand ED. The reagents may also include other specific binding proteins orsubstances which bind to analyte and analyte conjugated to ED,antibodies to analyte which compete with antibodies or antibodyfragments bound to ED for binding to analyte, antibodies to the specificbinding proteins or antibodies which increase the modulation of theassociation of EA and ED in response to analyte, or some combination ofreagents which modulate the generation of enzyme activity in response tothe presence of concentration of analyte. The EA and ED reagents may beprovided as mixtures of several active components, with other inactivecomponents (e.g., buffer salts and stabilizing proteins) added to theassay solution in various orders, with or without incubations betweenthe various additions.

[0024] The present invention also includes a substrate, which bears anelectroactive label, and an electrochemical cell. When EA, ED, and asample containing an analyte of interest are mixed, enzyme activity isgenerated in response to the presence or concentration of the analyte inthe sample mixture. The enzyme activity acts on the labeled substrate tofree label into the reaction mixture. The free label is then measuredelectrochemically by means known in the art. For example, the electrodesof the electrochemical cell may be maintained at a potential sufficientto cause oxidation of the freed label but not the label still bound tosubstrate. The current produced from the oxidation of the free label maythen be measured and correlated to the presence or amount of analyte inthe sample mixture.

Analytes

[0025] The presence or concentration of both high and low molecularweight analytes may be determined using the present invention. Examplesof such high molecular weight analytes include ferritin, hCG,carcinoembryonic antigen, human T-cell leukemia virus, insulin,α-fetoprotein, rubella virus, herpesvirus, cytomegalovirus, folliclestimulating hormone, thyroid stimulating hormone, leutinizing hormone,hepatitis virus, chorionic gonadotropin, estrogen receptor, thyroidstimulating hormone receptor, poliovirus receptor, insulin transportprotein, protein A, concanavalin A lectin, wheat germ aggulatininlectin, secretroy protein, cholera toxin, and avidin.

[0026] Examples of how molecular weight analytes include theophylline,vitamin B₁₂, folate, estriol, digozin, thryroxine, propranolol,methotrexate, phencyclidine, methadone, morphine, diazepam, oxazepam,quindine, propoxyphen, N-acetylprocainamide, secobarbital, tobramycin,gentamicin, amphetamine, benzoyl ecogonine, phenytoin, procainamide,lidocaine, carbamazepine, primidene, valproic acid, phenobarbital,ethosuximide, and biotin.

Enzyme Substrates

[0027] In the present invention, enzyme activity is monitoredelectrochemically by using an enzyme which is covalently linked to alabel. An example of such a labeled substrate is shown in FIG. 3.Labeled substrate 16(4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β-D-galactopyranoside) isnonelectroactive until enzymatic hydrolysis cleaves electroactive label17 (4-(1,4,7,10-tetraoxadecyl)-1-naphthol) from substrate 18(β-D-galactopyranose). Label 17 (4-(1,4,7,10-tetraoxadecyl)-1-naphthol)is then oxidized at the surface of an electrode, resulting in a currentwhich may be measured.

[0028] The β-galactosidase enzyme is especially suitable for use withthe present invention. Use of this enzyme results in smaller backgroundsignals and therefore greater analyte sensitivity, since there is nonatural β-galactosidase activity in human blood. β-galactosidasesubstrates that may be used with the present invention will now bedescribed. (If enzymes other than β-galactosidase are used in thepreparation of enzyme acceptor fragment EA and enzyme donor polypeptideconjugate ED, as described above, other substrates become necessary.)

[0029] In accordance with the present invention, the labeled substrateshould be soluble in aqueous medium and is preferably electrochemicallyinactive when scanned in the potential range of −0.6 V (volts) to +1.0 Vvs. Ag/AgCl. When cleaved from the substrate, the label should beelectrochemically active in this same potential range. Preferences forthe cleaved label include a near zero redox potential (0.0V<E°<0.5V vs.Ag/Ag Cl), electrochemical reversibility, and aqueous solubility.

[0030] Electrochemical characteristics of some β-galactosidase labeledsubstrates and their cleaved labels are provided in Table 1. TABLE 1Examples of β-galactosidase labeled substrates and their cleaved labelsCompound Elec. Active? E_(ox) vs. Ag/AGCL Reversible? Solubilityresorufin-β-D-galactopyranoside yes −0.100 V Yes soluble resorufin(cleaved) yes −0.100 V yes soluble 4-methoxy-1-naphthyl-β-D- yes −0.10V, +1.00 V quasi, no slightly galactopyranoside 4-methoxy-1-naphthol(cleaved) yes −0.10 V, +0 20 V quasi, no no p-aminophenyl-β-D- no — —soluble galactyopyranoside p-aminophenol (cleaved) yes +0 18 V yessoluble 4-(1, 4, 7, 10-tetraoxadecyl)-1- yes +0.05 V, +1.00 V no, nosoluble naphthyl-β-D-galactopyranoside 4-(1, 4, 7, 10-tetraoxadecyl)-1-yes +0 38 V no soluble naphthol (cleaved)

[0031] Although it is preferable for the labeled substrate to beelectrochemically inactive and the cleaved label electrochemicallyactive, both may be electrochemically active so long as they are activeat potentials at least 118 millivolts (mV) apart. (For example, labeledsubstrate 4-(1,4,7,10-oxadecyl)-1-naphthyl-β-D-galactopyranoside iselectrochemically active at +0.05 V and +1.00 V, but cleaved label4-(1,4,7,10-tetraoxadecyl)-1-naphthol is only electroactive at +0.38 V.)Referring to Table 1, labeled substrate4-(1,4,7,10-tetraoxadecyl)-β-D-naphthyl-β-D-galactopyranoside is thepreferred labeled substrate for the present invention because of itsaqueous solubility, which facilitates reagent formulation. Labeledsubstrates p-aminophenyl-β-D-galactopyranoside and4-methoxy-1-naphthyl-β-D -galactopyranoside also work well but are notpreferred since 4-methoxy-1-naphthyl-β-D-galactopyranoside is lesssoluble than 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β-D-galactopyranosideand p-aminophenyl-β-D-galactopyranoside has slower kinetics (i.e., thelabel is released more slowly under conditions of enzymatic hydrolysis)than 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β-D-galactopyranoside.Labeled substrate resorufin-β-D-galactopyranoside is an example of aβ-galactosidase labeled susbstrate that does not work with the presentinvention, since it is electrochemically active at the measurementpotential before and after cleavage.

[0032] Other labeled substrates which may be used to monitor enzymeactivity include 4-chloro-1-naphthyl-β-D-galactopyranoside,4-propoxy-1-naphthyl-β-D-galactopyranoside,4-isopropoxy-1-naphthyl-β-D-galactopyranoside,4-benzyloxy-1-naphthyl-β-D-galactopyranoside, and4-trifluoroethoxy-1-naphthyl-β-D-galactopyranoside. Preparation of theselabeled substrates and the labeled substrate4-methoxy-1-naphthyl-β-D-galactopyranoside (described above) isdisclosed in U.S. Pat. No. 5,202,233 (issued Apr. 13, 1993), thedisclosure of which is hereby incorporated by reference.

[0033] Other commercially-available labeled substrates which also may beused in accordance with the present invention includep-nitrophenyl-β-D-galactopyranoside, chlorphenolred-β-D-galactopyranoside (CPRG), o-nitrophenyl-β-D-galactopyranoside,umbelliferyl-β-D-galactopyranoside,o-methoxy-p-nitrophenyl-β-D-galactopyranoside,3,4-dinitrophenyl-β-D-galactopyrandoside,m-cyano-p-nitrophenyl-β-D-galactopyranoside,4-nitrosalicylaldehyde-β-D-galactopyranoside, and 4methyl-umbelliferyl-β-D-galactopyranoside.

[0034] The synthesis of 4-(1,4,7,10-tetraoxydecyl)-1-naphthol, disclosedby Goeltner et al., Liebigs Ann. Chem. 1991, 1085-1089, is as follows:2.0 grams (g) (125 millimoles (mmol)) naphthohydroquinone is added to 80milliliters (ml) triethylene glycol to yied 3.4 g (92%) of a violet oilwhich, after column chromatography (SO₂-saturated ethyl acetate),hardens into pink needles with a melting point of 70° C. The crudeproduct, 4-(1,4,7,10-tetraoxadecyl)-1-naphthol, is then attached toβ-D-galactopyranose to form labeled substrate4-(1,4,7,10-tetraoxadecyl)-1-naphthyl-β-D-galactopyranoside as describedbelow in the synthesis of 4-methoxy-1-naphthyl-β-D-galactopyranoside,except that 4-(1,4,7,10-tetraoxadecyl)-1-naphthol is used in place of4-methoxy-1-naphthol.

[0035] Methods of synthesis for p-aminophenyl-β-D-galactopyranoside andp-nitrophenyl-β-D-galactopyranoside are publicly known.

Electrochemical Cells

[0036] Performing an assay in accordance with the present inventioninvolves making an electrochemical measurement. One example of anelectrochemical cell that can be used to make such an electrochemicalmeasurement will now be described.

[0037] Reference is now made to FIGS. 4 & 5. Electrocell (e-cell) 19 hasa first insulating substrate 20, which is about 360 microns thick andmade of polyester. Other substrates and thicknesses could also be used.Typically, plastics such as vinyl polymers and polyimides provide theelectrical and structural properties which are desired. First electrode22 and second electrode 23 are each about 0.1 micron thick, made ofpalladium, and are affixed to first insulating substrate 20 by the useof hot melt adhesive (not shown). In addition to palladium, otherelectronically conducting materials may be used for electrodes 22 and23, including platinum, gold, silver, carbon, titanium, and copper.Noble metals and preferred because they provide a more constant,reproducible electrode surface area. Palladium is particularly preferredbecause it is one of the more difficult noble metals to oxide. Silver isnot preferred because it is more readily oxidized by air than by theother noble metals listed above. Electrodes 22 and 23 should besufficiently separated so that the electrochemical events at oneelectrode do not interfere with the electrochemical events at the otherelectrode.

[0038] Electrodes 22 and 23 are deposited on a backing of insulatormaterial 24, a polymide, to reduce the possibility of tearing theelectrode before it is affixed to substrate 20. Backing 24 is about 25microns thick. The electrode and polyimide combination is commerciallyavailable from Courtaulds Performance Films in California. Electrodes 22and 23 extend from one end of substrate 20 to the other end in parallelconfiguration. The distance between electrodes 22 and 23 is about 1.2 mm(millimeters).

[0039] Second insulating substrate 21 is fixed on top of firstinsulating substrate 20 and electrodes 22 and 23 by the use of hot meltadhesive (not shown). Substrate 21 is about 250 microns thick, made ofpolyester, and includes sample window 25 which exposes at least aportion of electrodes 22 and 23. Sample window 25 is 4 mm by 6 mm andelectrodes 22 and 23 are each 1.55 mm in width. Therefore, a surfacearea of about 6 mm² is exposed for each of the two electrodes. Substrate21 also has cutout portion 26 at one end to allow an electricalconnection between the electrodes and a power source (not shown) and acurrent measuring meter (not shown). As discussed above with substrate20, other substrates and thicknesses may be used for substrate 21.

[0040] In the electrocell embodiment described above, the first andsecond electrodes described are working and counter electrodes. Thisembodiment has the advantage of being easy to manufacture Although thedescribed embodiment only has the reduced form of the label attached tothe substrate, the oxidized form of the label (e.g.,4-(1,4,7,10-textraoxadecyl)-1-naphthal) should also preferably bepresent in the reagent in high concentration (at least twice theconcentration of the amount of the reduced form of the cleaved label(e.g., 4-(1,4,7,10-tetraoxadecyl)-1-naphthol) expected to be produced bythe assay) when using the working/counter electrode design describedabove. Since the oxidation of the label is being measured, the oxidationand not the reduction should be the current-limiting event. Having anexcess of the reduced form of the label helps ensure that the reductionof the label is not current-limiting.

[0041] Other electrocell configurations are possible. For example, a twoelectrode electrocell using a reference electrode (e.g., Ag/AgCl) ratherthan a counter electrode or a three electrode electrocell using working,counter and reference electrodes are possible. The preferred embodimentof the referenced two or three electrode electrocells would not need theoxidized form of the label present in the reagent.

EXAMPLE 1 Theophylline Assay

[0042] The Theophylline System Pack (a CEDIA® assay, commerciallyavailable from Boehringer Mannheim Corporation) is an EIA for thequantitative determination of theophylline in serum or plasma. TheTheophylline System Pack was modified and optimized to allowmeasurements to be performed in accordance with the present invention.The contents of the Theophylline System Pack referred to above will nowbe described.

[0043] The enzyme used in the Theophylline System Pack is split into twoinactive fragments, EA and ED, through the use of recombinant DNAtechnology. EA is a relatively large polypeptide containingapproximately 95% of the native β-galactosidase enzyme protein sequence.ED is a small polypeptide containing approximately 5% of the nativeβ-galactosidase enzyme. EA can spontaneously recombine with ED to form acatalytically active enzyme. The analyte analog is covalently bound toED in a way that does not interfere with reassociation of the enzymefragments

[0044] The Theophylline System Pack includes four primary components:(i) EA reagent (lyophilized), (ii) EA reconstitution buffer, (iii) EDreagent (lyophilized), and (iv) ED reconstitution bufer. The EA reagent(lyophilized) includes the EA fragment of the enzyme, monoclonalanti-theophylline antibody, buffer salts, surfactants, carrier proteins,and preservative. A vial of the EA reagent is reconstituted with 20 ml(milliliters) of EA reconstitution buffer. The EA reconstitution bufferincludes 3-N-morpholino)propanesulfonic acid buffer solution (MOPS),stabilizers, and preservatives.

[0045] The ED reagent (lyophilized) includes the ED fragment of theenzyme, buffer, chlorophenol-red-β-D-galactopyranoside (CPRG),surfactants, stabilizer, secondary antibody, and preservative. A vial ofthe ED reagent is reconstituted with 16 ml of ED reconstitution buffer,which is similar in composition to the EA reconstitution buffer.

[0046] The Theophylline System Pack described above may be modified foruse in the present invention. Referring to FIG. 2, analyte analog 1,enzyme donor polypeptide, 2, ED 3, antibody 4, EA 6, sample analyte 8,and active enzyme 10 all have the same function as in the CEDIA® assaydescribed in FIG. 1. However, labeled substrate 12 is made by covalentlylinking label 14 to substrate 13 in such a way that label 14 isnonelectroactive at the measurement potential until cleaved fromsubstrate 13 by enzymatic hydrolysis. Label 14 may then be oxidized atthe surface of electrode 15 to produce a current which may be correlatedto the detection or measurement of analyte 8 present in the sample beinganalyzed. The present invention allows an assay to be performed on asample of about 25 μl (microliters), whereas the Theophylline SystemPack assay requires a sample volume of about 250 μl.

[0047] The Theophylline System Pack was modified as described in Tables2 through 5, which identify the components, concentrations, andfunctions of the components in accordance with the present invention.TABLE 2 Theophylline System Pack modifications - ED reagent Modified EDReagent Concentration Function Potassium Phosphate, dibasic 80.000 mMbuffer Fragmented Bovine Serum Albumin 2.000 mg protects viability ofantibody and (BSA) (milligrams)/ml enzymes Heat inactivated goatanti-mouse serum 47.600 ml/l (liter) provides steric hindrance to (GAMS)antibody Labeled substrate (enzyme substrate) (see provides means formonitoring below) enzyme activity ED-theophylline conjugate 1.613 ml/lenzyme donor fragment - coupled to theophylline

[0048] TABLE 3 Theophylline System Pack modifications - EDreconstitution buffer Modified ED Reconstitution Buffer ConcentrationFunction Potassium Phosphate, dibasic 0.0205 M (molar) buffer PotassiumPhosphate, monobasic 0 0295 M buffer Sodium Chloride (NaCl) 1.00 Mprevents salting out of antibodies Tween 20 (10% aqueous solution)0.020% by volume surfactant

[0049] TABLE 4 Theophylline System Pack modifications - EA reagentModified EA Reagent Concentration Function Potassium Phosphate, dibasic4.000 mM (millimolar) buffer EA 203.520 U (Units)/ml enzyme acceptorfragment Theophylline monoclonal antibody 25 μg (micrograms)/mltheophylline antibody Theophylline high calibrator 40 mg/l shiftsreaction into linear range of curve

[0050] TABLE 5 Theophylline System Pack modifications - EAreconstitution buffer Modified EA Reconstitution Buffer ConcentrationFunction Potassium Phosphate, dibasic 0.0205 M buffer PotassiumPhosphate, monobasic 0.0295 M buffer Sodium Chloride (NaCl) 1.00 Mprevents salting out of antibodies Magnesium Acetate 0.0078 M providessource of Mg²⁺ for enzymatic reaction Tween 20 (10% aqueous solution)0.020% surfactant

[0051] The EA and ED reagents and reconstitution buffers described abovewere prepared as follows. EA reagent: a bulk potassium phosphate bufferwith a concentration of 4,000 mM was prepared in deionized, distilledwater. (Since an electrochemical measurement is being made in thepresent invention, it is important that the buffer is electrochemicallyinactive at the measurement potential.) The pH was adjusted to 7.1 at25° C. by addition of IN (normal) HCl (hydrochloric acid). The bufferwas then filtered through sterile 0 2 μm (micrometer) cellulose acetate.The enzyme acceptor fragment (EA) was cut using sodium sulfate and theEA concentration was titered before it was added to the bulk buffer atabout 200 U/ml (β-galactosidase units defined by its reaction withchlorophenol-red-β-D-galactopyranoside (CPRG), based on the extinctioncoefficient of the cleaved substrate). Theophylline monoclonal antibodywas added to the reagent at a concentration of about 25 μg/ml. Excesstheophylline was then added to the reagent at a concentration of 40mg/l. The excess theophylline increases the linearity of the system byshifting the low end of the calibration curve into the linear range. Thereagent was then assayed versus a reference reagent and adjusted toachieve appropriate activity (a titration to ensure there is enoughβ-galactosidase, theophylline, and theophylline monoclonal antibody tomeasure the highest concentration of theophylline to be detected by theassay). The reagent was then filtered through a 0.2 μm cellulose acetatefilter and 4 ml was lyophilized in a glass bottle.

[0052] ED reagent: a bulk potassium phosphate buffer with aconcentration of 80,000 mM was prepared in deionized, distilled water.(As stated above, it is important that the buffer is electrochemicallyinactive at the measurement potential.) Pepsin-digested BSA was thenadded to the buffer at a concentration of 2.0 mg/ml. The addition of BSA(protein fragments) to the ED reagent reduces hydrolysis of the enzymedonor polypeptide conjugate from proteases in the fluid sample (i.e.,the BSA enhances stability of the antibody and ED—the resultinghydrophobic interactions maintain the conformation of proteins) Thesynthesis of pepsin-digested BSA is described in example 2, column 7,lines 26-40 (using the 60 minute incubation period) of U.S. Pat. No.5,212,081.(Coty et al., issued May 18, 1993), the disclosure of which ishereby incorporated by reference. The pH was then adjusted to 7.1 at 25°C. Heat-inactivated goat anti-mouse serum (GAMS) was then added toachieve a protein concentration of 10 g (grams)/l. GAMS contains asecond antibody, capable of binding to theophylline monoclonal antibody,that helps reduce background signal by providing extra steric hindranceto ensure the ED fragment does not complement with the EA fragment whenED is bound to the monoclonal theophylline antibody. The reagent wasthen filtered through a 0.2 μm cellulose acetate filter. Finally, theenzyme donor polypeptide conjugate (ED) was added. The reagent was thenassayed versus a reference reagent, which contained the ED reagentcomponents in known concentrations. 4 ml of the ED reagent was thenlyophilized in a glass bottle.

[0053] EA reconstitution buffer: a potassium phosphate buffer wasprepared at a concentration of 0.05 M. Sodium chloride was added untilthe solution was 1.0 M in sodium chloride, and magnesium acetate wasadded until its concentration was 0.0078 M. A small amount of Tween 20detergent (see Table 5 above) was then added. The ED reconstitutionbuffer was prepared in the same manner, except that magnesium acetatewas not added.

[0054] Preparation of enzyme acceptor fragment EA and enzyme donorpolypeptide conjugate ED by recombinant DNA methods in accordance withthe present invention is fully described in U.S. Pat. No. 4,708,929,incorporated by reference above.

[0055] An assay for theophylline was then performed as follows. Alabeled substrate stock solution was prepared which included 0.307 molar(M) 4-methoxy-1-naphthyl-β-D-galactopyranoside in DMSO. 20 ml of the EAreconstitution buffer was added to the lyophilized EA reagent. 15.877 mlof the ED reconstitution buffer and 0.123 ml of the labeled substratestock solution was added to the lyophilized ED reagent. 293 μl(microliters) of the reconstituted EA reagent was dispensed into anincubated tube. 23 μl of a serum sample was then added to thereconstituted EA reagent and the solution was briefly and gently mixed.The reconstituted EA reagent/sample mixture was allowed to incubate at atemperature of 35-37° C. for 4 minutes and 36 seconds. 220 μl of thereconstituted ED reagent was then added to the reconstituted EAreagent/sample mixture and the solution was briefly and gently mixed.The full mixture was then allowed to incubate at a temperature of 35-37°C. for 19 minutes and 16 seconds.

[0056] After the final incubation, about 20 μl of the full mixture wasapplied to the sample window of the electrocell described above andshown in FIGS. 4 and 5. The electrodes were electrically connected to apower source and a current measuring meter. When palladium first andsecond electrodes are utilized in the electrocell, a potentialdifference of 450 mV (millivolts) was applied between the twoelectrodes. (Potential differences less than 450 mV are not preferredbecause non-diffusion-limited currents are possible. Potentialdifferences greater than 450 mV are not preferred because unnecessaryoxidation of interfering compounds in the sample is possible.) Thecurrent generated was measured for about 5 seconds. The amount ofcurrent measured 3 seconds after application of the potential differencewas then compared to a calibration curve and theophylline concentrationin the serum sample was determined. (Current reading times of less than3 seconds are not preferred because less precise measurements mayresult. Current reading times of greater than 3 seconds are notpreferred because smaller currents and lower sensitivity may result.)Minimal background signals were observed, since there is noβ-galactosidase activity in human blood, the uncombined EA and EDfragments are not electrochemically active, and because there are fewendogenous electrochemically active compounds in blood. As a result,practice of the present invention results in enhanced analytesensitivity

EXAMPLE 2 Theophylline Assay Using a Dry-Chemistry Immunosensor

[0057] In addition to using the aqueous reagents and electrocelldescribed above, the present invention could also be practiced by usinga dry-chemistry immunosensor. Two examples of such an immunosensor willnow be described.

[0058] Reference is now made to immunosensor 28 shown in FIGS. 6 & 7.First insulating substrate 29, first electrode 31, second electrode 32,and insulator material 33 are all similar in composition and function tofirst insulating substrate 20, first electrode 22, second electrode 23,and insulator material 24 described above in FIGS. 4 & 5. (Atwo-electrode electrocell and a three-electrode electrocell, utilizingworking and reference electrodes, are also possible, as described abovefor electrocell 19.) Immunosensor 28 also has second insulatingsubstrate 30, fixed on top of first insulating substrate 29 andelectrodes 31 and 32 by the use of hot melt adhesive (not shown).Substrate 30 is about 250 microns thick, made of polyester, and includeswindow 35 which exposes at least a portion of electrodes 31 and 32.Substrate 30 also has cutout portion 34 at one end to allow anelectrical connection between the electrodes and a power source (notshown) and a current measuring meter (not shown).

[0059] Immunosensor 28 also has a polyester mesh 36. Polyester mesh 36may be any porous substrate that has sufficient porosity to allowpassage of a whole blood sample. Examples of porous substrates that maybe used include meshes, films, soluble polymers, and membranes Polyestermesh 36 is impregnated with the ED reagent (described above) bydispensing about 5 μl of the ED reagent directly onto mesh 36. Mesh 36is then dried by heating at about 50° C. for about 15 minutes. After thereagent has dried, mesh 36 is affixed above window 35 in secondinsulating substrate 30 as shown in FIG. 7.

[0060] About 6 μl of EA reagent 37 (described above) is dispenseddirectly onto second insulating substrate 30 as shown in FIG. 7. Thirdinsulating substrate 38 is placed over second insulating substrate 30.Third insulating substrate 38 is a thin insulating substrate whichpreferably has adhesive material on each side to hold it in place. Thirdinsulating substrate 38 includes cutout portion 41. Fourth insulatingsubstrate 39 (not shown in FIG. 6) is placed over third insulatingsubstrate 38, such that a capillary space is formed within cutoutportion 41 of third insulating substrate 38 which allows capillary flowfrom EA reagent 37 to polyester mesh 36 (which is impregnated with EDreagent). Fourth insulating substrate 39 is about 250 microns thick,made of polyester, and includes sample window 40 (not shown in FIG. 6)which exposes EA reagent 37, and vent hole 42 (not shown in FIG. 6).

[0061] Immunosensor 28 may be used to determine the concentration of ananalyte in a whole blood sample by the following method. Whole bloodsample 43 (about 20 μl) is applied to sample window 40 of immunosensor28. A mixture of EA reagent 37 and blood sample 43 is formed, which isdrawn to polyester mesh 36 by capillary action caused by cutout portion41 and vent 42. The ED reagent, impregnated in mesh 36, then becomespart of the mixture. The mixture of EA reagent 37, ED reagent, and bloodsample 43 then settles on electrodes 31 and 32 through window 35 ofsecond insulating substrate 30. After an incubation period of about 20minutes, a potential difference of 450 mV is applied between electrodes31 and 32 (palladium first and second electrodes, as described above,electrically connected to a power source and a current measuring meter).The current generated is measured for about 5 seconds. The amount ofcurrent measured 3 seconds after application of the potential differenceis then compared to a calibration curve and analyte concentration in thewhole blood sample is determined.

EXAMPLE 3 Theophylline Assay Using a Dry-Chemistry Immunosensor

[0062] Another example of a dry chemistry immunosensor that can be usedto practice the present invention will now be described. Immunosensor 44shown in FIGS. 8 & 9 includes first insulating substrate 45, firstelectrode 47, second electrode 48, insulator material 49, secondinsulating substrate 46, cutout portion 50, and window 51, which are allsimilar in composition and function to first insulating substrate 29,first electrode 31, second electrode 32, insulator material 33, secondinsulating substrate 30, cutout portion 34, and window 35 describedabove in FIGS. 6 & 7. (A two-electrode electrocell, utilizing workingand reference electrodes, and a three-electrode electrocell are alsopossible, as described above for electrocell 19 and immunosensor 28.)

[0063] About 5 μL of ED reagent 52 and 6 μL of EA reagent 53 aredispensed directly onto second insulating substrate 46 as shown in FIGS.8 & 9. ED reagent 52 and EA reagent 53 are made as described above,dispensed onto second insulating substrate 46, then dried by heating atabout 50° C. for about 15 minutes. Second insulating substrate 46 alsohas polymer 54, placed between ED reagent 52 and EA reagent 53. Polymer54 can be any water-soluble polymer, such as polyvinyl pyridine,polyvinyl pyrrolidone, or polyvinyl imidazole, and preferably should benon-electroactive and non-reactive.

[0064] Third insulating substrate 55 is placed over second insulatingsubstrate 46. Third insulating substrate 55 is a thin insulatingsubstrate which preferably has adhesive material on each side to hold itin place. Third insulating substrate 55 includes cutout portion 58.Fourth insulating substrate 56 (not shown in FIG. 8) is placed overthird insulating substrate 55, such that capillary space is formedwithin cutout portion 58 of third insulating substrate 55 which allowscapillary flow from EA reagent 53 to ED reagent 52. Fourth insulatingsubstrate 56 is about 250 microns thick, made of polyester, and includessample window 57 (not shown in FIG. 8) which exposes EA reagent 53, andvent hole 59 (not shown in FIG. 8).

[0065] Immunosensor 44 may be used to determine the concentration of ananalyte in a whole blood sample by the following method. Whole bloodsample 60 (about 20 μl) is applied to sample window 57 of immunosensor44 A mixture of EA reagent 53 and blood sample 60 is formed. Polymer 54allows whole blood sample 60 and EA reagent 53 to mix and react for apredetermined period of time, during which time polymer 54 is dissolved.After polymer 54 has dissolved, the solution of whole blood sample 60,EA reagent 53 and polymer 54 then flows to ED reagent 52 to completemixing the immunoassay reaction components. The EA reagent 53, EDreagent 52, polymer 54, and blood sample 60 mixture is then drawn bycapillary action (caused by cutout portion 58 and vent hole 59) towindow 51 and settles on electrodes 47 and 48. After an incubationperiod of about 20 minutes, a potential difference of 450 mV is appliedbetween electrodes 47 and 48 (palladium first and second electrodes, asdescribed above, electrically connected to a power source and a currentmeasuring meter). The current generated is measured for about 5 seconds.The amount of current measured 3 seconds after application of thepotential difference is then compared to a calibration curve and analyteconcentration in the whole blood sample is determined.

[0066] The meter and power source described above for use withelectrocell 19 and immunosensors 28 and 44 will normally be adapted toapply an algorithm to the current measurement, whereby the presence orconcentration of analyte is provided and visually displayed.Improvements in such a power source, meter, and biosensor system are thesubject of commonly assigned U.S. Pat. No. 4,963,814 (issued Oct. 16,1990), U.S. Pat. No. 4,999,632 (issued Mar. 12, 1991), U.S. Pat. No.4,999,582 (issued Mar. 12, 1991), U.S. Pat. No. 5,243,516 (issued Sep.7, 1993), U.S. Pat. No. 5,352,351 (issued Oct. 4, 1994), U.S. Pat. No.5,366,609 (issued Nov. 22, 1994), White et al., U.S. Pat. No. 5,405,511,issued Apr. 11, 1995 and White at al., U.S. Pat. No. 5,438,271, issuedAug. 1, 1995, the disclosures of which are hereby incorporated byreference.

EXAMPLE 4 Ferritin Assay

[0067] The Theophylline System Pack described above in Example 1 mayalso be modified as described below in order to perform an assay forferritin according to the present invention.

[0068] In the ED reagent, goat antibody to ferritin coupled to ED isused at 0.4 nanomolar (nM) concentration in place of the theophylline-EDconjugate. The goat antibody may be coupled to ED through any number ofstandard techniques for protein crosslinking. For example, the goatantibody may be reacted with maleimide hexyl succinimide (MHS) to supplymaleimide groups, and the ED is coupled to the activated goat antibodythrough the cysteine residues present. The product is then purified withgel filtration chromatography. (See, for example, “Chemistry of ProteinConjugation”, Shan W. Wong, CRC Press.) In the EA reagent, mousemonoclonal antibody to ferritin is used at a concentration of 187 μg/mlin place of the mouse monoclonal antibody to theophylline. The ED and EAbuffers remain the same, since they contain no theophylline-specificcomponents.

[0069] A ferritin assay may be carried out as follows. 20 ml of EDbuffer is added to the lyophilized ED reagent and allowed to dissolve(solution 1). 16 ml of EA buffer is added to lyophilized EA reagent andallowed to dissolve (solution 2). 35 μl of a sample containing ferritinis mixed with 100 μl of solution 1. This mixture is incubated for 12minutes. 80 μl of solution 2 is then added to the mixture. The mixtureis then allowed to incubate an additional 4 minutes. A 20 μl sample ofthis mixture is then applied to an electrochemical cell and ameasurement made as described above. The current measured is thenrelated to the concentration of ferritin in the sample throughcalibration standards. In this assay, the amount of current measured isinversely proportional to the amount of ferritin in the sample.

EXAMPLE 5 hCG Assay

[0070] The Theophylline System Pack described above in Example 1 mayalso be modified as described below in order to perform an assay for hCGaccording to the present invention.

[0071] The ED reagent contains the non-specific components listed abovein Example 1. Antibody to hCG coupled to ED is used at 3.2 nM in placeof the theophylline-ED conjugate. Goat antibody to biotin is alsoincluded at a concentration of 80 μg/ml. The EA reagent also containsthe non-theophylline-specific components listed in Example 1, plusdonkey antiserum to goat antibodies (added after a 13-fold dilution ofthe heat-treated serum). The ED and EA buffers remain the same as shownin Example 1. The hCG assay also requires a capture reagent whichincludes hCG conjugated to biotin. This conjugate may be prepared bystandard techniques for coupling haptens to proteins (e g., addingbiotin-NHS ester (Pierce) dissolved in DMF to a solution of hCG incarbonate buffer, pH 9.0, incubating 1 hour, and removing unreactedbiotin by gel filtration with a PD-10 column (Pharmacia)). The capturereagent contains the hCG biotin dissolved at 35 nM in EA buffer.

[0072] An hCG assay may be carried out as follows. 20 ml of ED buffer isadded to the lyophilized ED reagent and allowed to dissolve (solution1). 16 ml of EA buffer is added to lyophilized EA reagent and allowed todissolve (solution 2). 35 μl of a sample containing hCG is mixed with 50μl of solution 1 and incubated for 5 minutes. 75 μl capture reagent isthen added, followed by an additional 5 minute incubation. 105 μlsolution 2 is then added to the mixture and incubated for an additional2.5 minutes. About 20 μl of the reaction mixture is then applied to anelectrochemical cell and a measurement made as described above. Thecurrent measured is related to the sample hCG concentration through astandard curve. In this assay the current measured is directlyproportional to the hCG concentration in the sample.

EXAMPLE 6 Vitamin B₁₂ Assay

[0073] The B₁₂ CEDIA® assay, commercially available from BoehringerMannheim Corporation, may be modified as described below in order toperform an assay for B₁₂ according to the present invention. Themodification to the B₁₂ CEDIA® assay would be carried out in a similarmanner to the Theophylline System Pack modification described above inExample 1.

[0074] The ED reagent, which contains ED conjugated to cyanocobolamine(B₁₂ analog), is modified by using an electrochemically labeledsubstrate in place of the colorimetically labeled substrate provided inthe commercial kit. The EA reagent, ED and EA buffers, pre-treatmentreagent, and binding protein reagent remain unchanged from thecommercial kit.

[0075] A B₁₂ assay may be carried out as follows. ED reagent isreconstituted with 15 ml ED buffer to form solution 1. EA reagent isreconstituted with 15 ml EA buffer to form solution 2 The bindingprotein reagent is reconstituted with 17 ml ED buffer. 48 μl of a samplecontaining B₁₂ is mixed with 57 μl of pre-treatment reagent andincubated 80 seconds. 125 μl of the binding protein reagent is added andincubated an additional 215 seconds. 100 μl of solution 1 is added andincubated an additional 375 seconds. 100 μl of solution 2 is added andincubated an additional 590 seconds. About 20 μl is then removed fromthe reaction mixture and placed in an electrochemical cell and ameasurement made as described above. The current measured is related tothe sample B₁₂ concentration through a standard curve.

EXAMPLE 7 Folate Assay

[0076] The folate CEDIA® assay, commercially available from BoehringerMannheim Corporation, may be modified as described below in order toperform an assay for folate according to the present invention. Themodification to the folate CEDIA® assay would be carried out in asimilar manner to the Theophylline System Pack modification describedabove in Example 1.

[0077] The ED reagent, which contains ED bound to pteroylglutamic acid,is modified by using an electrochemically labeled substrate in place ofthe colorimetically labeled substrate provided in the commercial kit.The EA reagent, ED and EA buffers, pre-treatment reagent, and folatebinding protein are unmodified from the commercial kit.

[0078] A folate assay may be carried out as follows. ED reagent isreconstituted with 15 ml ED buffer to form solution 1. EA reagent isreconstituted with 15 ml EA buffer to form solution 2. The folatebinding protein is reconstituted with 15 ml ED buffer to form a bindingprotein solution. 42 μl of a sample containing folate is added to 42 μlof pre-treatment reagent and incubated 80 seconds. 100 μl of the bindingprotein solution is added and incubated an additional 215 seconds. 100μl of solution 1 is added and incubated an additional 395 seconds. 100μl of solution 2 is added and incubated an additional 590 seconds. About20 μl is then removed from the reaction mixture and placed in anelectrochemical cell and a measurement made as described above. Thecurrent measured is related to the sample folate concentration through astandard curve.

[0079] The present invention has been disclosed in the above teachingsand drawings with sufficient clarity and conciseness to enable oneskilled in the art to make and use the invention, to know the best modefor carrying out the invention, and to distinguish it from otherinventions and from what is old. Many variations and obvious adaptationsof the invention will readily come to mind, and these are intended to becontained within the scope of the invention as claimed below.

What is claimed is:
 1. A diagnostic kit for determining, in a directmanner, the presence or concentration of an analyte in a fluid sample,wherein the diagnostic kit utilizes an electrochemical measurement,comprising: (a) an enzyme donor reagent which comprises 1) an enzymedonor polypeptide conjugate comprising an enzyme donor polypeptideconjugated to an antibody; and 2) a labeled substrate, comprising anenzyme substrate cleavably linked to an electroactive label, and (b) anenzyme acceptor reagent which comprises an enzyme acceptor polypeptidecapable of combining with the enzyme donor polypeptide conjugate to forman active enzyme complex capable of catalyzing the cleavage of theelectroactive label from the substrate, wherein the amount of activeenzyme complex formed is directly related to the amount of analyte inthe fluid sample.
 2. The diagnostic kit of claim 1, wherein the enzymesubstrate comprises 13-D-galactopyranoside.
 3. The diagnostic kit ofclaim 1, wherein the electroactive label comprises at least one of4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl,4-chloro-1-naphthyl, 4-propoxy-1-naphthyl, 4-isopropoxy-1-naphthyl,4-benzyloxy-1-naphthyl, 4-trifluoroethoxy-1-naphthyl, p-nitrophenyl,chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl,3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or4-methyl-umelliferyl.
 4. The diagnostic kit of claim 1, wherein theenzyme acceptor reagent further comprises a first antibody capable ofimmunologically binding to the analyte.
 5. An electrochemicalimmunoassay method for determining the presence or concentration of ananalyte in a fluid sample, comprising: (a) preparing a mixture whichincludes; (1) the fluid sample; (2) an enzyme donor reagent, whichcomprises (i) an enzyme donor polypeptide conjugate comprising an enzymedonor polypeptide conjugated to an antibody; and (ii) a labeledsubstrate, comprising an enzyme substrate cleavably linked to anelectroactive label; and (3) an enzyme acceptor reagent, which comprisesan enzyme acceptor polypeptide capable of combining with the enzymedonor polypeptide conjugate to form an amount of an active enzymecomplex capable of catalyzing the cleavage of the substrate from thesubstrate; (b) applying the mixture to an electrochemical cell havingfirst and second electrodes; (c) applying, after incubation of themixture, a potential difference between the first and second electrodessufficient to oxidize the electroactive label that has been cleaved fromthe substrate, thereby generating a current; and (d) measuring thecurrent and correlating the current to the presence or concentration ofthe analyte, wherein the amount of active enzyme complex formed isdirectly related to the amount of analyte in the fluid sample.
 6. Theelectrochemical immunoassay method of claim 5, wherein the enzymesubstrate comprises β-D-galactopyranoside.
 7. The electrochemicalimmunoassay method of claim 5, wherein the electroactive label comprisesat least one of 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl,4-methoxy-1-naphthyl, 4-chloro-1-naphthyl, 4-propoxy-1-naphthyl,4-isopropoxy-1-naphthyl, 4-benzyloxy-1-naphthyl,4-trifluoroethoxy-1-naphthyl, p-nitrophenyl, chlorophenol red,o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl,m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.8. The electrochemical immunoassay method of claim 5, wherein the enzymeacceptor reagent further comprises a first antibody capable ofimmunologically binding to the analyte.
 9. An immunosensor useful in adirect electrochemical immunoassay of an analyte in a fluid sample,comprising: (a) a first insulating substrate; (b) first and secondelectrodes affixed to the first insulating substrate; (c) a secondinsulating substrate, which overlays the first and second electrodes,has a window for exposing at least a portion of the first and secondelectrodes, and has a cutout portion at one end to allow contact betweenthe electrodes and a meter and a power source; (d) an enzyme donorreagent which is placed on the second insulating substrate, the enzymedonor reagent comprising (1) an enzyme donor polypeptide conjugatecomprising an enzyme donor polypeptide conjugated to an antibody; and(2) a labeled substrate, comprising an enzyme substrate cleavably linkedto an electroactive label; (e) an enzyme acceptor reagent, which isplaced on the second insulating substrate, the enzyme acceptor reagentcomprising an enzyme acceptor polypeptide capable of combining with theenzyme donor polypeptide conjugate to form an active enzyme complexcapable of catalyzing the cleavage of the from the substrate wherein theamount of active enzyme complex formed is directly related to the amountof analyte in the fluid sample; and (f) a polymer, which is placed onthe second insulating substrate between the enzyme acceptor reagent andthe enzyme donor reagent; (g) a third insulating substrate, whichoverlays the second insulating substrate and has a cutout portion forexposing the enzyme donor reagent, the enzyme acceptor reagent, thepolymer, and the window in the second insulating substrate; and (h) afourth insulating substrate, which overlays the third insulatingsubstrate such that a capillary space is formed within the cutoutportion of the third insulating substrate, has a window for exposing aportion of the enzyme acceptor reagent, and has a vent hole.
 10. Theimmunosensor of claim 9, wherein the enzyme substrate comprisesβ-D-galactopyranoside.
 11. The immunosensor of claim 9, wherein theelectroactive label comprises at least one of4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl,4-chloro-1-naphthyl, 4-propoxy-1-naphthyl, 4-isopropoxy-1-naphthyl,4-benzyloxy-1-naphthyl, 4-trifluoroethoxy-1-naphthyl, p-nitrophenyl,chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl,3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or4-methyl-umelliferyl.
 12. The immunosensor of claim 9, wherein theenzyme acceptor reagent further comprises a first antibody capable ofimmunologically binding to the analyte.